Although the toxic nature of many chemicals, such as carcinogenic species, which have found their way into the environment is well-known, the mechanisms by which these chemicals exert their toxic effects at the cellular and molecular level are only now beginning to be understood. There is a strong need for developing a molecular tool that is capable of monitoring biochemical processes or detecting specific biomarkers in a living cell following chemical exposure. Minimally invasive analysis of cellular signaling pathways inside single intact cells is becoming increasingly important fundamentally because cells in a population respond asynchronously to external stimuli. The objective of this research is to conduct fundamental research and develop nanobiosensors for probing chemical exposure and health effects of individual living cells. The proposed technique could provide unprecedented insights into intact cell function, allowing studies of molecular functions in the context of the functional cell architecture. This proposed research will contribute to future advancements in biology and medicine, which is directly relevant to the mission of National Institute of Environmental Health Sciences (NIEHS). The specific aims of the project are: 1) Develop a nanosensor for monitoring the carcinogenic compound, benzo[a]pyrene (BaP) and the related biomarker of exposure, benzopyrene tetrol (BPT), 2) Develop dual-target sensing technology for the nanobiosensor to achieve simultaneous detection of BaP and BPT using phase-resolved detection, and 3) Investigation of exposure and metabolism pathways in a single living cell exposed to BaP. The nanoprobes to be developed in this project will open new horizons to a host of applications in biotechnology, molecular biology and environmental health research, and the study of in situ intracellular signaling processes, and investigations of the chemical transport and toxicity.